Natriuretic polypeptides

ABSTRACT

This document provides methods and materials related to natriuretic polypeptides and the use of natriuretic polypeptides to treat cardiovascular and/or renal conditions. For example, chimeric polypeptides having at least one amino acid segment (e.g., N-terminus tail, ring structure, C-terminus tail, or a combination thereof) of a natriuretic peptide (e.g., ANP, BNP, CNP, URO, or DNP) and an amino acid segment of an angiotensin polypeptide (e.g., Ang-(1-7)) are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/003,481, filed Sep. 6, 2013, which is a National Stage applicationunder 35 U.S.C. §371 of International Application No. PCT/US2012/051734,filed Aug. 21, 2012, which claims the benefit of U.S. ProvisionalApplication Ser. No. 61/648,718, filed May 18, 2012 and U.S. ProvisionalApplication Ser. No. 61/529,113, filed Aug. 30, 2011. The disclosures ofthe prior applications are considered part of (and are incorporated byreference in) the disclosure of this application.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersHL036634 and HL076611 awarded by National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND

1. Technical Field

This document relates to natriuretic polypeptides. For example, thisdocument provides methods and materials related to natriureticpolypeptides and the use of natriuretic polypeptides to treatcardiovascular and renal conditions.

2. Background Information

Natriuretic polypeptides are polypeptides that can cause natriuresis(increased sodium excretion in the urine). Such polypeptides can beproduced by brain, heart, kidney, and/or vascular tissue. Thenatriuretic peptide family in humans includes the cardiac hormonesatrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP),C-type natriuretic peptide (CNP), and urodilatin (URO). Natriureticpolypeptides function via guanylyl cyclase receptors (i.e., NPR-A forANP, BNP, and URO; and NPR-B for CNP) and the second messenger cyclic3′5′ guanosine monophosphate (cGMP) (Kuhn, Circ. Res., 93:700-709(2003); Tawaragi et al., Biochem. Biophys. Res. Commun., 175:645-651(1991); and Komatsu et al., Endocrinol., 129:1104-1106 (1991)).

SUMMARY

This document provides methods and materials related to natriureticpolypeptides and the use of natriuretic polypeptides to treatcardiovascular and/or renal conditions. For example, this documentprovides chimeric polypeptides having at least one amino acid segment(e.g., N-terminus tail, ring structure, C-terminus tail, or acombination thereof) of a natriuretic peptide (e.g., ANP, BNP, CNP, URO,or Dendroaspis natriuretic peptide (DNP)) and an amino acid segment ofan angiotensin polypeptide (e.g., Ang-(1-7)).

As described herein, a chimeric polypeptide can be designed to includethe Ang-(1-7) amino acid sequence attached as the C terminus of the ringstructure of CNP in a manner that results in a chimeric polypeptidehaving the ability to stimulate human cardiac fibroblasts to producecGMP. These results demonstrate that chimeric polypeptides can bedesigned to include an amino acid segment of an angiotensin polypeptide(e.g., Ang-(1-7)) and at least one amino acid segment (e.g., N-terminustail, tail, or a combination thereof) of a natriuretic peptide (e.g.,ANP, BNP, CNP, URO, or Dendroaspis natriuretic peptide (DNP)) in amanner that allows the chimeric polypeptide to exhibit an activity suchas the ability to activate cGMP production. In some cases, a chimericpolypeptide provided herein can exhibit a diuretic activity, anatriuretic activity, the ability to activate cGMP, the ability toincrease glomerular filtration rate, the ability to reduce reninproduction, the ability to reduce angiotensin production, the ability toreduce aldosterone production, the ability to reduce abnormally elevatedcardiac filling pressures, the ability to optimize renal blood flow, ora combination thereof. In some cases, a chimeric polypeptide providedherein can be a chimeric natriuretic polypeptide.

In general, one aspect of this document features a polypeptide from 17to 50 amino acid residues in length. The polypeptide comprises, orconsists essentially of, in an order from amino terminus to carboxyterminus, (a) the sequence set forth in SEQ ID NO:2 or the sequence setforth in SEQ ID NO:2 with no more than three additions, subtractions, orsubstitutions, (b) the sequence set forth in SEQ ID NO:3 or the sequenceset forth in SEQ ID NO:3 with no more than five additions, subtractions,or substitutions, and (c) the sequence set forth in SEQ ID NO:1 or thesequence set forth in SEQ ID NO:1 with no more than two additions,subtractions, or substitutions. The polypeptide can have acGMP-activating property. The polypeptide can comprise the sequence setforth in SEQ ID NO:2. The polypeptide can comprise the sequence setforth in SEQ ID NO:3. The polypeptide can comprise the sequence setforth in SEQ ID NO:1.

In another aspect, this document features a polypeptide from 17 to 50amino acid residues in length. The polypeptide comprises, or consistsessentially of, in an order from amino terminus to carboxy terminus, (a)the sequence set forth in SEQ ID NO:4 or the sequence set forth in SEQID NO:4 with no more than three additions, subtractions, orsubstitutions, (b) the sequence set forth in SEQ ID NO:5 or the sequenceset forth in SEQ ID NO:5 with no more than five additions, subtractions,or substitutions, and (c) the sequence set forth in SEQ ID NO:1 or thesequence set forth in SEQ ID NO:1 with no more than two additions,subtractions, or substitutions. The polypeptide can have acGMP-activating property. The polypeptide can comprise the sequence setforth in SEQ ID NO:4. The polypeptide can comprise the sequence setforth in SEQ ID NO:5. The polypeptide can comprise the sequence setforth in SEQ ID NO:1.

In another aspect, this document features a polypeptide from 17 to 50amino acid residues in length. The polypeptide comprises, or consistsessentially of, in an order from amino terminus to carboxy terminus, (a)the sequence set forth in SEQ ID NO:6 or the sequence set forth in SEQID NO:6 with no more than three additions, subtractions, orsubstitutions, (b) the sequence set forth in SEQ ID NO:7 or the sequenceset forth in SEQ ID NO:7 with no more than five additions, subtractions,or substitutions, and (c) the sequence set forth in SEQ ID NO:1 or thesequence set forth in SEQ ID NO:1 with no more than two additions,subtractions, or substitutions. The polypeptide can have acGMP-activating property. The polypeptide can comprise the sequence setforth in SEQ ID NO:6. The polypeptide can comprise the sequence setforth in SEQ ID NO:7. The polypeptide can comprise the sequence setforth in SEQ ID NO:1.

In another aspect, this document features a polypeptide from 17 to 50amino acid residues in length. The polypeptide comprises, or consistsessentially of, in an order from amino terminus to carboxy terminus, (a)the sequence set forth in SEQ ID NO:8 or the sequence set forth in SEQID NO:8 with no more than three additions, subtractions, orsubstitutions, (b) the sequence set forth in SEQ ID NO:9 or the sequenceset forth in SEQ ID NO:9 with no more than five additions, subtractions,or substitutions, and (c) the sequence set forth in SEQ ID NO:1 or thesequence set forth in SEQ ID NO:1 with no more than two additions,subtractions, or substitutions. The polypeptide can have acGMP-activating property. The polypeptide can comprise the sequence setforth in SEQ ID NO:8. The polypeptide can comprise the sequence setforth in SEQ ID NO:9. The polypeptide can comprise the sequence setforth in SEQ ID NO:1.

In another aspect, this document features a polypeptide from 17 to 50amino acid residues in length. The polypeptide comprises, or consistsessentially of, in an order from amino terminus to carboxy terminus, (a)the sequence set forth in SEQ ID NO:10 or the sequence set forth in SEQID NO:10 with no more than three additions, subtractions, orsubstitutions, (b) the sequence set forth in SEQ ID NO:11 or thesequence set forth in SEQ ID NO:11 with no more than five additions,subtractions, or substitutions, and (c) the sequence set forth in SEQ IDNO:1 or the sequence set forth in SEQ ID NO:1 with no more than twoadditions, subtractions, or substitutions. The polypeptide can have acGMP-activating property. The polypeptide can comprise the sequence setforth in SEQ ID NO:10. The polypeptide can comprise the sequence setforth in SEQ ID NO:11. The polypeptide can comprise the sequence setforth in SEQ ID NO:1.

In another aspect, this document features a polypeptide from 17 to 50amino acid residues in length. The polypeptide comprises, or consistsessentially of, in an order from amino terminus to carboxy terminus, (a)the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQID NO:1 with no more than two additions, subtractions, or substitutions,(b) the sequence set forth in SEQ ID NO:2 or the sequence set forth inSEQ ID NO:2 with no more than five additions, subtractions, orsubstitutions, and (c) the sequence set forth in SEQ ID NO:12 or thesequence set forth in SEQ ID NO:12 with no more than two additions,subtractions, or substitutions. The polypeptide can have acGMP-activating property. The polypeptide can comprise the sequence setforth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:2. The polypeptide can comprise the sequence setforth in SEQ ID NO:12.

In another aspect, this document features a polypeptide from 17 to 50amino acid residues in length. The polypeptide comprises, or consistsessentially of, in an order from amino terminus to carboxy terminus, (a)the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQID NO:1 with no more than two additions, subtractions, or substitutions,(b) the sequence set forth in SEQ ID NO:5 or the sequence set forth inSEQ ID NO:5 with no more than five additions, subtractions, orsubstitutions, and (c) the sequence set forth in SEQ ID NO:13 or thesequence set forth in SEQ ID NO:13 with no more than two additions,subtractions, or substitutions. The polypeptide can have acGMP-activating property. The polypeptide can comprise the sequence setforth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:5. The polypeptide can comprise the sequence setforth in SEQ ID NO:13.

In another aspect, this document features a polypeptide from 10 to 14amino acid residues in length. The polypeptide comprises, or consistsessentially of, in an order from amino terminus to carboxy terminus, (a)the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQID NO:1 with no more than two additions, subtractions, or substitutions,and (b) the sequence set forth in SEQ ID NO:12 or the sequence set forthin SEQ ID NO:12 with no more than two additions, subtractions, orsubstitutions. The polypeptide can comprise a cGMP-activating property.The polypeptide can comprise the sequence set forth in SEQ ID NO:1. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:12. Thepolypeptide can lack a ring structure.

In another aspect, this document features a polypeptide from 10 to 16amino acid residues in length. The polypeptide comprises, or consistsessentially of, in an order from amino terminus to carboxy terminus, (a)the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQID NO:1 with no more than two additions, subtractions, or substitutions,and (b) the sequence set forth in SEQ ID NO:13 or the sequence set forthin SEQ ID NO:13 with no more than two additions, subtractions, orsubstitutions. The polypeptide can comprise a cGMP-activating property.The polypeptide can comprise the sequence set forth in SEQ ID NO:1. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:13. Thepolypeptide can lack a ring structure.

In another aspect, this document features a polypeptide from 19 to 25amino acid residues in length. The polypeptide comprises, or consistsessentially of, in an order from amino terminus to carboxy terminus, (a)the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQID NO:1 with no more than two additions, subtractions, or substitutions,and (b) the sequence set forth in SEQ ID NO:30 or the sequence set forthin SEQ ID NO:30 with no more than five additions, subtractions, orsubstitutions. The polypeptide can comprise a cGMP-activating property.The polypeptide can comprise the sequence set forth in SEQ ID NO:1. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:30. Thepolypeptide can lack a ring structure.

In another aspect, this document features a polypeptide from 10 to 14amino acid residues in length. The polypeptide comprises, or consistsessentially of, in an order from amino terminus to carboxy terminus, (a)the sequence set forth in SEQ ID NO:12 or the sequence set forth in SEQID NO:12 with no more than two additions, subtractions, orsubstitutions, and (b) the sequence set forth in SEQ ID NO:1 or thesequence set forth in SEQ ID NO:1 with no more than two additions,subtractions, or substitutions. The polypeptide can comprise acGMP-activating property. The polypeptide can comprise the sequence setforth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:12. The polypeptide can lack a ring structure.

In another aspect, this document features a polypeptide from 10 to 16amino acid residues in length. The polypeptide comprises, or consistsessentially of, in an order from amino terminus to carboxy terminus, (a)the sequence set forth in SEQ ID NO:13 or the sequence set forth in SEQID NO:13 with no more than two additions, subtractions, orsubstitutions, and (b) the sequence set forth in SEQ ID NO:1 or thesequence set forth in SEQ ID NO:1 with no more than two additions,subtractions, or substitutions. The polypeptide can comprise acGMP-activating property. The polypeptide can comprise the sequence setforth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:13. The polypeptide can lack a ring structure.

In another aspect, this document features a polypeptide from 19 to 25amino acid residues in length. The polypeptide comprises, or consistsessentially of, in an order from amino terminus to carboxy terminus, (a)the sequence set forth in SEQ ID NO:30 or the sequence set forth in SEQID NO:30 with no more than five additions, subtractions, orsubstitutions, and (b) the sequence set forth in SEQ ID NO:1 or thesequence set forth in SEQ ID NO:1 with no more than two additions,subtractions, or substitutions. The polypeptide can comprise acGMP-activating property. The polypeptide can comprise the sequence setforth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:30. The polypeptide can lack a ring structure.

In another aspect, this document features a polypeptide from 20 to 28amino acid residues in length. The polypeptide comprises, in an orderfrom amino terminus to carboxy terminus (a) the sequence set forth inSEQ ID NO:1 or the sequence set forth in SEQ ID NO:1 with no more thantwo additions, subtractions, or substitutions, and (b) the sequence setforth in SEQ ID NO:7 or the sequence set forth in SEQ ID NO:7 with nomore than five additions, subtractions, or substitutions. Thepolypeptide can comprise a cGMP-activating property. The polypeptide cancomprise the sequence set forth in SEQ ID NO:1. The polypeptide cancomprise the sequence set forth in SEQ ID NO:7. The polypeptide can lacka C-terminal tail attached to a ring structure.

In another aspect, this document features a polypeptide from 20 to 28amino acid residues in length. The polypeptide comprises, or consistsessentially of, in an order from amino terminus to carboxy terminus, (a)the sequence set forth in SEQ ID NO:36 or the sequence set forth in SEQID NO:36 with no more than five additions, subtractions, orsubstitutions, and (b) the sequence set forth in SEQ ID NO:1 or thesequence set forth in SEQ ID NO:1 with no more than two additions,subtractions, or substitutions. The polypeptide can comprise acGMP-activating property. The polypeptide can comprise the sequence setforth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:36. The polypeptide can lack a N-terminal tailattached to a ring structure.

In another aspect, this document features a polypeptide from 20 to 28amino acid residues in length. The polypeptide comprises, or consistsessentially of, in an order from amino terminus to carboxy terminus, (a)the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQID NO:1 with no more than two additions, subtractions, or substitutions,and (b) the sequence set forth in SEQ ID NO:36 or the sequence set forthin SEQ ID NO:36 with no more than five additions, subtractions, orsubstitutions. The polypeptide can comprise a cGMP-activating property.The polypeptide can comprise the sequence set forth in SEQ ID NO:1. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:36. Thepolypeptide can lack a C-terminal tail attached to a ring structure.

In another aspect, this document features a polypeptide from 20 to 28amino acid residues in length. The polypeptide comprises, or consistsessentially of, in an order from amino terminus to carboxy terminus, (a)the sequence set forth in SEQ ID NO:7 or the sequence set forth in SEQID NO:7 with no more than five additions, subtractions, orsubstitutions, and (b) the sequence set forth in SEQ ID NO:1 or thesequence set forth in SEQ ID NO:1 with no more than two additions,subtractions, or substitutions. The polypeptide can comprise acGMP-activating property. The polypeptide can comprise the sequence setforth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:7. The polypeptide can lack a N-terminal tailattached to a ring structure.

In another aspect, this document features a polypeptide from 17 to 50amino acid residues in length. The polypeptide comprises, or consistsessentially of, in an order from amino terminus to carboxy terminus, (a)the sequence set forth in SEQ ID NO:2, the sequence set forth in SEQ IDNO:4, the sequence set forth in SEQ ID NO:6, the sequence set forth inSEQ ID NO:8, the sequence set forth in SEQ ID NO:10, the sequence setforth in SEQ ID NO:2 with no more than three additions, subtractions, orsubstitutions, the sequence set forth in SEQ ID NO:4 with no more thanthree additions, subtractions, or substitutions, the sequence set forthin SEQ ID NO:6 with no more than three additions, subtractions, orsubstitutions, the sequence set forth in SEQ ID NO:8 with no more thanthree additions, subtractions, or substitutions, or the sequence setforth in SEQ ID NO:10 with no more than three additions, subtractions,or substitutions, (b) the sequence set forth in SEQ ID NO:3, thesequence set forth in SEQ ID NO:5, the sequence set forth in SEQ IDNO:7, the sequence set forth in SEQ ID NO:9, the sequence set forth inSEQ ID NO:11, the sequence set forth in SEQ ID NO:3 with no more thanfive additions, subtractions, or substitutions, the sequence set forthin SEQ ID NO:5 with no more than five additions, subtractions, orsubstitutions, the sequence set forth in SEQ ID NO:7 with no more thanfive additions, subtractions, or substitutions, the sequence set forthin SEQ ID NO:9 with no more than five additions, subtractions, orsubstitutions, or the sequence set forth in SEQ ID NO:11 with no morethan five additions, subtractions, or substitutions, and (c) thesequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ IDNO:1 with no more than two additions, subtractions, or substitutions.The polypeptide can comprise a cGMP-activating property. The polypeptidecan comprise the sequence set forth in SEQ ID NO:2, 4, 6, 8, or 10. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:3, 5, 7, 9,or 11. The polypeptide can comprise the sequence set forth in SEQ IDNO:1. The polypeptide can comprise the sequence set forth in SEQ IDNO:43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59with no more than five additions, subtractions, or substitutions. Thepolypeptide can be from 17 to 45 amino acid residues, from 17 to 40amino acid residues, from 17 to 35 amino acid residues, from 20 to 50amino acid residues, from 25 to 50 amino acid residues, from 20 to 45amino acid residues, from 20 to 40 amino acid residues, from 20 to 35amino acid residues, from 25 to 45 amino acid residues, from 25 to 40amino acid residues, or from 25 to 35 amino acid residues, in length.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a structural schematic of a chimeric polypeptide containing anN-terminus and ring structure of a natriuretic peptide and a C-terminusof a segment of an angiotensin polypeptide in accordance with someembodiments. The amino acid segment of an angiotensin polypeptide shownin FIG. 1 (DRVYIHP; SEQ ID NO:1) can be referred to as angiotensin-(1-7)or Ang-(1-7). The N-terminus and ring structure can have the sequence ofany appropriate natriuretic peptide including, without limitation, ANP,BNP, CNP, DNP, and URO. “A” refers to an amino acid sequence from theN-terminus of a natriuretic peptide, “B” refers to an amino acidsequence from the ring structure of a natriuretic peptide, and “C”refers to an amino acid sequence of Ang(1-7), which can form theC-terminus of a chimeric polypeptide.

FIG. 2 is a structural schematic of a chimeric polypeptide (SEQ IDNO:43) containing an N-terminus and ring structure of ANP and aC-terminal Ang-(1-7) in accordance with some embodiments. The amino acidsequence of the N-terminal segment of ANP shown in FIG. 2 (SLRRSS; SEQID NO:2) can be referred to as ANP_(N-term), while the amino acidsequence of the ring structure segment of ANP shown in FIG. 2(CFGGRMDRIGAQ-SGLGC; SEQ ID NO:3) can be referred to as ANP_(ring).

FIG. 3 is a structural schematic of a chimeric polypeptide (SEQ IDNO:44) containing an N-terminus and ring structure of BNP and aC-terminal Ang-(1-7) in accordance with some embodiments. The amino acidsequence of the N-terminal segment of BNP shown in FIG. 3 (SPKMVQGSG;SEQ ID NO:4) can be referred to as BNP_(N-term), while the amino acidsequence of the ring structure segment of BNP shown in FIG. 3(CFGRKM-DRISSSSGLGC; SEQ ID NO:5) can be referred to as BNP_(ring). Thechimeric polypeptide having the amino acid sequence set forth in SEQ IDNO:44 can be referred to as BNP-Ang1-7.

FIG. 4 is a structural schematic of a chimeric polypeptide (SEQ IDNO:45) containing an N-terminus and ring structure of CNP and aC-terminal Ang-(1-7) in accordance with some embodiments. The amino acidsequence of the N-terminal segment of CNP shown in FIG. 4 (GLSKG; SEQ IDNO:6) can be referred to as CNP_(N-term), while the amino acid sequenceof the ring structure segment of CNP shown in FIG. 4(CFGLKLDRIG-SMSGLGC; SEQ ID NO:7) can be referred to as CNP_(ring). Thechimeric polypeptide shown in FIG. 4 can be referred to as cAng or cANG.

FIG. 5 is a structural schematic of a chimeric polypeptide (SEQ IDNO:46) containing an N-terminus and ring structure of DNP and aC-terminal Ang-(1-7) in accordance with some embodiments. The amino acidsequence of the N-terminal segment of DNP shown in FIG. 5 (EVKYDP; SEQID NO:8) can be referred to as DNP_(N-term), while the amino acidsequence of the ring structure segment of DNP shown in FIG. 5(CFGHKIDRINHVS-NLGC; SEQ ID NO:9) can be referred to as DNP_(ring).

FIG. 6 is a structural schematic of a chimeric polypeptide (SEQ IDNO:47) containing an N-terminus and ring structure of URO and aC-terminal Ang-(1-7) in accordance with some embodiments. The amino acidsequence of the N-terminal segment of URO shown in FIG. 6 (TAPRSLRRSS;SEQ ID NO:10) can be referred to as URO_(N-term), while the amino acidsequence of the ring structure segment of URO shown in FIG. 6(CFGG-RMDRIGAQSGLGC; SEQ ID NO:11) can be referred to as URO_(ring).

FIG. 7 is a structural schematic of a chimeric polypeptide (SEQ IDNO:48) containing an N-terminus of BNP (BNP_(N-term)), a ring structureof ANP (ANP_(ring)), and a C-terminal Ang-(1-7) in accordance with someembodiments.

FIG. 8 is a structural schematic of a chimeric polypeptide (SEQ IDNO:49) containing an N-terminus of CNP (CNP_(N-term)), a ring structureof ANP (ANP_(ring)), and a C-terminal Ang-(1-7) in accordance with someembodiments.

FIG. 9 is a structural schematic of a chimeric polypeptide (SEQ IDNO:50) containing an N-terminus of DNP (DNP_(N-term)), a ring structureof ANP (ANP_(ring)), and a C-terminal Ang-(1-7) in accordance with someembodiments.

FIG. 10 is a structural schematic of a chimeric polypeptide (SEQ IDNO:51) containing an N-terminus of URO (URO_(N-term)), a ring structureof ANP (ANP_(ring)), and a C-terminal Ang-(1-7) in accordance with someembodiments.

FIG. 11 is a structural schematic of a chimeric polypeptide (SEQ IDNO:52) containing an N-terminus of ANP (ANP_(N-term)), a ring structureof BNP (BNP_(ring)), and a C-terminal Ang-(1-7) in accordance with someembodiments.

FIG. 12 is a structural schematic of a chimeric polypeptide (SEQ IDNO:53) containing an N-terminus of CNP (CNP_(N-term)), a ring structureof BNP (BNP_(ring)), and a C-terminal Ang-(1-7) in accordance with someembodiments.

FIG. 13 is a structural schematic of a chimeric polypeptide (SEQ IDNO:54) containing an N-terminus of DNP (DNP_(N-term)), a ring structureof BNP (BNP_(ring)), and a C-terminal Ang-(1-7) in accordance with someembodiments.

FIG. 14 is a structural schematic of a chimeric polypeptide (SEQ IDNO:55) containing an N-terminus of URO (URO_(N-term)), a ring structureof BNP (BNP_(ring)), and a C-terminal Ang-(1-7) in accordance with someembodiments.

FIG. 15 is a structural schematic of a chimeric polypeptide (SEQ IDNO:56) containing an N-terminus of ANP (ANP_(N-term)), a ring structureof CNP (CNP_(ring)), and a C-terminal Ang-(1-7) in accordance with someembodiments.

FIG. 16 is a structural schematic of a chimeric polypeptide (SEQ IDNO:57) containing an N-terminus of BNP (BNP_(N-term)), a ring structureof CNP (CNP_(ring)), and a C-terminal Ang-(1-7) in accordance with someembodiments.

FIG. 17 is a structural schematic of a chimeric polypeptide (SEQ IDNO:58) containing an N-terminus of DNP (DNP_(N-term)), a ring structureof CNP (CNP_(ring)), and a C-terminal Ang-(1-7) in accordance with someembodiments.

FIG. 18 is a structural schematic of a chimeric polypeptide (SEQ IDNO:59) containing an N-terminus of URO (URO_(N-term)), a ring structureof CNP (CNP_(ring)), and a C-terminal Ang-(1-7) in accordance with someembodiments.

FIG. 19 is a structural schematic of a chimeric polypeptide (SEQ IDNO:60) containing an N-terminal Ang-(1-7) and a ring structure(ANP_(ring)) and C-terminus of ANP in accordance with some embodiments.The amino acid sequence of the C-terminal segment of ANP shown in FIG.19 (NSFRY; SEQ ID NO:12) can be referred to as ANP_(C-term).

FIG. 20 is a structural schematic of a chimeric polypeptide (SEQ IDNO:61) containing an N-terminal Ang-(1-7) and a ring structure(BNP_(ring)) and C-terminus of BNP in accordance with some embodiments.The amino acid sequence of the C-terminal segment of BNP shown in FIG.20 (KVLRRH; SEQ ID NO:13) can be referred to as BNP_(C-term). Thechimeric polypeptide having the amino acid sequence set forth in SEQ IDNO:61 can be referred to as Ang1-7BNP.

FIG. 21 is a graph plotting the level of cGMP (pmol/mL) produced byhuman cardiac fibroblasts when exposed to ANP (10⁻⁶M), BNP (10⁻⁶M), CNP(10⁻⁶M), or cANG (10⁻⁶, 10⁻⁸, or 10⁻¹⁰ M).

FIG. 22 is a graph plotting the level of cGMP (pmol/mL) produced byhuman cardiac fibroblasts when exposed to ANP (10⁻⁶ M), BNP (10⁻⁶ M),CNP (10⁻⁶ M), or cANG (10⁻⁶M) either alone or in the presence of A-71915(1 μM; an NPR-A blocker (see, e.g., Kumar et al., Hypertension, 29(1 Pt2):414-21 (1997))) or an NPR-B antibody (1:100 dilution).

FIG. 23 is a structural schematic of a chimeric polypeptide (SEQ IDNO:27) containing an N-terminal Ang-(1-7) and the C-terminus of ANP(ANP_(C-term)) without a ring structure in accordance with someembodiments. This polypeptide can be referred to as Ang-(1-7)-ANP-CT.

FIG. 24 is a structural schematic of a chimeric polypeptide (SEQ IDNO:28) containing an N-terminal Ang-(1-7) and the C-terminus of BNP(BNP_(C-term)) without a ring structure in accordance with someembodiments. This polypeptide can be referred to as Ang-(1-7)-BNP-CT.

FIG. 25 is a structural schematic of a chimeric polypeptide (SEQ IDNO:29) containing an N-terminal Ang-(1-7) and the C-terminus of DNP(DNP_(C-term)) without a ring structure in accordance with someembodiments. This polypeptide can be referred to as Ang-(1-7)-DNP-CT.The amino acid sequence of the C-terminal segment of DNP shown in FIG.25 (PSLRDPRPNAPSTSA; SEQ ID NO:30) can be referred to as DNP_(C-term).

FIG. 26 is a structural schematic of a chimeric polypeptide (SEQ IDNO:31) containing the C-terminus of ANP (ANP_(C-term)) as an N-terminalsegment followed by a C-terminal Ang-(1-7) without a ring structure inaccordance with some embodiments. This polypeptide can be referred to asANP-CT-Ang-(1-7).

FIG. 27 is a structural schematic of a chimeric polypeptide (SEQ IDNO:32) containing the C-terminus of BNP (BNP_(C-term)) as an N-terminalsegment followed by a C-terminal Ang-(1-7) without a ring structure inaccordance with some embodiments. This polypeptide can be referred to asBNP-CT-Ang-(1-7).

FIG. 28 is a structural schematic of a chimeric polypeptide (SEQ IDNO:33) containing the C-terminus of DNP (DNP_(C-term)) as an N-terminalsegment followed by a C-terminal Ang-(1-7) without a ring structure inaccordance with some embodiments. This polypeptide can be referred to asDNP-CT-Ang-(1-7).

FIG. 29 is a structural schematic of a chimeric polypeptide (SEQ IDNO:34) containing an N-terminal Ang-(1-7) and a ring structure(CNP_(ring)) with no C-terminal tail in accordance with someembodiments. This polypeptide can be referred to as Ang-(1-7)-CNPR1.

FIG. 30 is a structural schematic of a chimeric polypeptide (SEQ IDNO:35) containing a reverse ring structure of CNP and an C-terminalAng-(1-7) and with no N-terminal tail in accordance with someembodiments. This polypeptide can be referred to as Ang-(1-7)-CNPR2. Theamino acid sequence of the reverse ring structure segment of CNP shownin FIG. 30 (CGLGSMSGIRDLKLGFC; SEQ ID NO:36) can be referred to asreverse-CNP_(ring).

FIG. 31 is a structural schematic of a chimeric polypeptide (SEQ IDNO:37) containing an N-terminal Ang-(1-7) and a reverse ring structure(reverse-CNP_(ring)) with no C-terminal tail in accordance with someembodiments. This polypeptide can be referred to as Ang-(1-7)-CNPR3.

FIG. 32 is a structural schematic of a chimeric polypeptide (SEQ IDNO:38) containing a ring structure (CNP_(ring)) and an C-terminalAng-(1-7) and with no N-terminal tail in accordance with someembodiments. This polypeptide can be referred to as Ang-(1-7)-CNPR4.

FIG. 33 contains graphs plotting cGMP levels resulting from treatment ofHEK 293 cells with the indicated amount (10⁻⁶, 10⁻⁸, or 10⁻¹⁰ M) of BNP(BNP1-32; FIG. 33A), Ang1-7BNP (FIG. 33B), or BNP-Ang1-7 (FIG. 33C). TheHEK 293 cells were stably transfected to express NPR-A (i.e., GC-A).

FIG. 34 contains graphs plotting cGMP levels resulting from treatment ofHEK 293 cells with the indicated amount (10⁻⁶, 10⁻⁸, or 10⁻¹⁰ M) of BNP(BNP1-32; FIG. 34A), Ang1-7BNP (FIG. 34B), or BNP-Ang1-7 (FIG. 34C). TheHEK 293 cells were stably transfected to express NPR-B (i.e., GC-B).

FIG. 35 contains graphs plotting cGMP levels resulting from treatment ofHEK 293 cells with the indicated amount (10⁻⁶, 10⁻⁸, or 10⁻¹⁰ M) ofAng1-7. The HEK 293 cells were stably transfected to express NPR-A(i.e., GC-A) (left graph) or NPR-B (i.e., GC-B) (right graph).

DETAILED DESCRIPTION

This document provides methods and materials related to natriureticpolypeptides and the use of natriuretic polypeptides to treatcardiovascular and/or renal conditions. For example, this documentprovides chimeric polypeptides having at least one amino acid segment(e.g., N-terminus tail, ring structure, reverse ring structure,C-terminus tail, or a combination thereof) of a natriuretic peptide(e.g., ANP, BNP, CNP, URO, or DNP) and an amino acid segment of anangiotensin polypeptide (e.g., Ang-(1-7), Ang-(1-8), or Ang-(1-9)). Insome cases, a chimeric polypeptide provided herein can be used toincrease natriuretic activity in a subject in need thereof. In somecases, a chimeric polypeptide provided herein can be used to increaseplasma cGMP levels, urinary cGMP excretion, net renal cGMP generation,urine flow, urinary sodium excretion, urinary potassium excretion,hematocrit, plasma BNP immunoreactivity, renal blood flow, and/or plasmaANP immunoreactivity. In some cases, a chimeric polypeptide providedherein can be used to decrease renal vascular resistance, proximal anddistal fractional reabsorption of sodium, mean arterial pressure,pulmonary capillary wedge pressure, right atrial pressure, pulmonaryarterial pressure, plasma renin activity, plasma angiotensin II levels,plasma aldosterone levels, renal perfusion pressure, and/or systemicvascular resistance. In some cases, a chimeric polypeptide providedherein can be used to treat, inhibit, and/or prevent cardiac remodelingand ischemia-reperfusion injury, particularly after acute myocardialinfarction (AMI) and/or acute heart failure (AHF). For example, achimeric polypeptide provided herein can be used to increase plasmacGMP, which may be desirable for applications in attenuating myocardialischemia-reperfusion injury (Padilla et al., Cardiovasc. Res.,51:592-600 (2001)).

As described herein, a chimeric polypeptide can be designed to includeat least one amino acid segment (e.g., N-terminus tail, ring structure,reverse ring structure, C-terminus tail, or a combination thereof) of anatriuretic peptide and an amino acid segment of an angiotensinpolypeptide. Examples of natriuretic peptides or “NPs” include, withoutlimitation, ANP, BNP, CNP, URO, and DNP. A chimeric polypeptide providedherein can include any appropriate amino acid segment of an angiotensinpolypeptide (e.g., human angiotensin polypeptide). For example, achimeric polypeptide provided herein can include the sequence set forthin SEQ ID NO:1. In some cases, a chimeric polypeptide provided hereincan include a full length angiotensin polypeptide (e.g., a full lengthhuman angiotensin polypeptide). For example, a chimeric polypeptideprovided herein can include the following sequence: DRVYIHPFHL (SEQ IDNO:14).

A chimeric polypeptide provided herein can include a ring structure of anatriuretic peptide. Examples of ring structures include, withoutlimitation, ANP_(ring), BNP_(ring), CNP_(ring), DNP_(ring), andURO_(ring). In some cases, an ANP_(ring), BNP_(ring), CNP_(ring),DNP_(ring), or URO_(ring) having one or more (e.g., one, two, three,four, five, six, or more) amino acid additions, subtractions, orsubstitutions can be used. For example, an ANP_(ring) or BNP_(ring)having two amino acid substitutions can be used as a ring structure of achimeric polypeptide provided herein.

In some cases, a chimeric polypeptide provided herein includes a reversering structure of a natriuretic peptide. Examples of reverse ringstructures include, without limitation, reverse-ANP_(ring)(CGLGSQAGIRDMRGGFC; SEQ ID NO:39), reverse-BNP_(ring)(CGLGSSSSIRDMKRGFC; SEQ ID NO:40), reverse-CNP_(ring),reverse-DNP_(ring) (CGLNSVHNIRDIKHGFC; SEQ ID NO:41), andreverse-URO_(ring) (CGLGSQAGIRDMRGGFC; SEQ ID NO:42). In some cases, areverse-ANP_(ring), reverse-BNP_(ring), reverse-CNP_(ring),reverse-DNP_(ring), or reverse-URO_(ring) having one or more (e.g., one,two, three, four, five, six, or more) amino acid additions,subtractions, or substitutions can be used. For example, areverse-ANP_(ring) or reverse-BNP_(ring) having two amino acidsubstitutions can be used as a ring structure of a chimeric polypeptideprovided herein.

In some cases, a chimeric polypeptide provided herein can include anyappropriate amino acid segment of an angiotensin polypeptide either asan N-terminal portion or as a C-terminal portion with respect to a ringstructure or reverse ring structure for those polypeptides containingsuch a ring structure or reverse ring structure. For example, a chimericpolypeptide provided herein can include an amino acid segment of anangiotensin polypeptide (e.g., Ang-(1-7)) followed by a ring structureand optionally a C-terminus of a natriuretic peptide (e.g.,ANP_(C-term), BNP_(C-term), or DNP_(C-term)). In some cases, an optionalN-terminus of a natriuretic peptide (e.g., ANP_(N-term) or BNP_(N-term))can be followed by a ring structure of a natriuretic peptide and anamino acid segment of an angiotensin polypeptide (e.g., Ang-(1-7)).

In some cases, a chimeric polypeptide provided herein can include anyappropriate amino acid segment of an angiotensin polypeptide either asan N-terminal portion or as a C-terminal portion attached to anN-terminus (e.g., ANP_(N-term), BNP_(N-term), CNP_(N-term),DNP_(N-term), or URO_(N-term)) or C-terminus of a natriuretic peptide(e.g., ANP_(C-term), BNP_(C-term), or DNP_(C-term)) without a ring orreverse ring structure. For example, a chimeric polypeptide providedherein can include an amino acid segment of an angiotensin polypeptide(e.g., Ang-(1-7)) followed by a C-terminus of a natriuretic peptide(e.g., ANP_(C-term) or BNP_(C-term)) without a ring or reverse ringstructure. In some cases, a C-terminus of a natriuretic peptide (e.g.,ANP_(C-term) or BNP_(C-term)) can be followed by an amino acid segmentof an angiotensin polypeptide (e.g., Ang-(1-7)) without a ring orreverse ring structure.

With reference to FIG. 1, a chimeric polypeptide provided herein caninclude an N-terminus and ring structure of a natriuretic peptide and aC-terminal Ang-(1-7). In some cases, the N-terminus and ring structurecan be of the same natriuretic peptide (see, e.g., FIGS. 2-6) or ofdifferent natriuretic peptides (see, e.g., FIG. 7-18). For example, achimeric polypeptide provided herein can have BNP_(N-term) followed byBNP_(ring) followed by Ang-(1-7) as shown in FIG. 3 or can haveANP_(N-term) followed by BNP_(ring) followed by Ang-(1-7) as shown inFIG. 11. In some cases, a chimeric polypeptide provided herein caninclude an N-terminal Ang-(1-7) followed by a ring structure andC-terminus of a natriuretic peptide as shown in FIGS. 19 and 20.

In some cases, an N-terminus, ring structure, reverse ring structure,and/or C-terminus of an NP included in a chimeric polypeptide providedherein can include a variant (e.g., a substitution, addition, ordeletion) at one or more positions (e.g., one, two, three, four, five,six, seven, eight, nine, or ten positions). Such variant NP sequences,e.g., those having one or more amino acid substitutions relative to anative NP amino acid sequence, can be prepared and modified as describedherein. Amino acid substitutions can be made, in some cases, byselecting substitutions that do not differ significantly in their effecton maintaining (a) the structure of the peptide backbone in the area ofthe substitution, (b) the charge or hydrophobicity of the molecule atthe target site, or (c) the bulk of the side chain. For example,naturally occurring residues can be divided into groups based onside-chain properties: (1) hydrophobic amino acids (norleucine,methionine, alanine, valine, leucine, and isoleucine); (2) neutralhydrophilic amino acids (cysteine, serine, and threonine); (3) acidicamino acids (aspartic acid and glutamic acid); (4) basic amino acids(asparagine, glutamine, histidine, lysine, and arginine); (5) aminoacids that influence chain orientation (glycine and proline); and (6)aromatic amino acids (tryptophan, tyrosine, and phenylalanine)Substitutions made within these groups can be considered conservativesubstitutions. Non-limiting examples of useful substitutions include,without limitation, substitution of valine for alanine, lysine forarginine, glutamine for asparagine, glutamic acid for aspartic acid,serine for cysteine, asparagine for glutamine, aspartic acid forglutamic acid, proline for glycine, arginine for histidine, leucine forisoleucine, isoleucine for leucine, arginine for lysine, leucine formethionine, leucine for phenyalanine, glycine for proline, threonine forserine, serine for threonine, tyrosine for tryptophan, phenylalanine fortyrosine, and/or leucine for valine.

Examples of variant N-terminal portions of NP sequences that can be usedto make a chimeric polypeptide provided herein include, withoutlimitation, SAPRSLRRSS (SEQ ID NO:15), TVPRSLRRSS (SEQ ID NO:16),TAGRSLRRSS (SEQ ID NO:17), TAPKSLRRSS (SEQ ID NO:18), TLRRSS (SEQ IDNO:19), SIRRSS (SEQ ID NO:20), SLKRSS (SEQ ID NO:21), and SLRKSS (SEQ IDNO:22). Examples of variant C-terminal portions of NP sequences that canbe used to make a chimeric polypeptide provided herein include, withoutlimitation, KVLRRR (SEQ ID NO:23), KVLRKH (SEQ ID NO:24), KVLKRH (SEQ IDNO:25), and KVIRRH (SEQ ID NO:26).

Further examples of conservative substitutions that can be made at anyposition within an NP amino acid sequence used to make a chimericpolypeptide provided herein include, without limitation, those set forthin Table 1.

TABLE 1 Examples of conservative amino acid substitutions. OriginalResidue Exemplary substitutions Ala Val, Leu, Ile Arg Lys, Gln, Asn AsnGln, His, Lys, Arg Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln,Lys, Arg Ile Leu, Val, Met, Ala, Phe, Norleucine Leu Norleucine, Ile,Val, Met, Ala, Phe Lys Arg, Gln, Asn Met Leu, Phe, Ile Phe Leu, Val,Ile, Ala Pro Gly Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe, Thr, Ser Val Ile,Leu, Met, Phe, Ala, Norleucine

In some cases, an NP amino acid sequence used to make a chimericpolypeptide provided herein can include one or more non-conservativesubstitutions. Non-conservative substitutions typically entailexchanging a member of one of the classes described above for a memberof another class. Such production can be desirable to provide largequantities or alternative embodiments of such compounds. Whether anamino acid change results in a functional polypeptide can readily bedetermined by assaying the specific activity of the peptide variant.

A chimeric polypeptide provided herein can have any appropriatesequence. For example, a polypeptide can include the sequences set forthin SEQ ID NOs:2, 3, and 1. In some cases, a chimeric polypeptideprovided herein can contain (a) an amino acid sequence that aligns tothe sequence set forth in SEQ ID NO:1 with three or less (e.g., two orless, one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof and (b) an amino acid sequence that aligns to thesequence of ANP_(ring), BNP_(ring), CNP_(ring), DNP_(ring), orURO_(ring) with five or less (e.g., four or less, three or less, two orless, one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof. In some cases, a chimeric polypeptide providedherein can contain (a) an amino acid sequence that aligns to thesequence set forth in SEQ ID NO:1 with three or less (e.g., two or less,one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof followed by (b) an amino acid sequence that alignsto the sequence of ANP_(ring), BNP_(ring), CNP_(ring), DNP_(ring), orURO_(ring) with five or less (e.g., four or less, three or less, two orless, one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof followed by (c) an amino acid sequence that alignsto the sequence of ANP_(C-term), BNP_(C-term), or a DNP_(C-term)sequence with five or less (e.g., four or less, three or less, two orless, one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof. In some cases, a chimeric polypeptide providedherein can contain (a) an amino acid sequence that aligns to thesequence of ANP_(N-term), BNP_(N-term), CNP_(N-term), DNP_(N-term), orURO_(N-term) with five or less (e.g., four or less, three or less, twoor less, one, or zero) amino acid additions, deletions, substitutions,or combinations thereof followed by (b) an amino acid sequence thataligns to the sequence of ANP_(ring), BNP_(ring), CNP_(ring),DNP_(ring), or URO_(ring) with five or less (e.g., four or less, threeor less, two or less, one, or zero) amino acid additions, deletions,substitutions, or combinations thereof followed by an amino acidsequence that aligns to the sequence set forth in SEQ ID NO:1 with threeor less (e.g., two or less, one, or zero) amino acid additions,deletions, substitutions, or combinations.

A polypeptide provided herein can have any appropriate length. Forexample, a polypeptide provided herein can be between 20 and 55 (e.g.,between 24 and 55, between 24 and 45, between 25 and 45, between 26 and44, between 27 and 43, between 28 and 42, between 29 and 41, between 30and 40, between 31 and 39, between 23 and 35, between 25 and 30, orbetween 30 and 35) amino acid residues in length. It will be appreciatedthat a polypeptide with a length of 20 or 55 amino acid residues is apolypeptide with a length between 20 and 55 amino acid residues.

Chimeric polypeptides provided herein as well as polypeptides containinga variant NP sequence with conservative and/or non-conservativesubstitutions (e.g., with respect to a natural ANP, BNP, CNP, DNP, orURO), fragments of ANP, BNP, CNP, DNP, or URO, or fragments of suchvariants can be assessed for biological activity using any suitableassay including, without limitation, those described herein. Forexample, the activity of a chimeric polypeptide having a variant NPamino acid sequence as described herein can be evaluated in vitro bymeasuring its effect on cGMP levels generated by cardiac fibroblasts(CFs) or by testing its ability to suppress proliferation of CFs. Suchexperiments can be performed, for example, in human CFs (ScienCell, SanDiego, Calif.) as described elsewhere (Tsuruda et al., Circ. Res.91:1127-1134 (2002)). Cells can be exposed to a polypeptide to beassessed (e.g., 10⁻¹¹ to 10⁻⁶ M), and samples can be assayed for cGMPusing a competitive RIA cGMP kit (Perkin-Elmer, Boston, Mass.). For CFproliferation studies, cells can be treated with Cardiotrophin-1 toinduce cell proliferation. A polypeptide to be assessed can be added tothe Cardiotrophin-1-stimulated CFs to determine its effect on cellproliferation. Cell proliferation can be detected and measured using,for example, a colormetric bromodeoxyuridine (BrdU) cell proliferationELISA (Roche, Indianapolis, Ind.).

In some cases, a chimeric polypeptide provided herein, a polypeptidecontaining a variant NP sequence with conservative and/ornon-conservative substitutions (e.g., with respect to a natural ANP,BNP, CNP, DNP, or URO), a fragment of ANP, BNP, CNP, DNP, or URO, or afragment of such variants can be assessed in vivo by, for example,testing its effects on factors such as pulmonary capillary wedgepressure, right atrial pressure, mean arterial pressure, urinary sodiumexcretion, urine flow, proximal and distal fractional sodiumreabsorption, plasma renin activity, plasma cGMP levels, urinary cGMPexcretion, net renal generation of cGMP, glomerular filtration rate, andleft ventricular mass in animals. In some cases, such parameters can beevaluated after induced myocardial infarction (e.g., myocardialinfarction induced by coronary artery ligation).

In some embodiments, a chimeric polypeptide provided herein can becyclic due to disulfide bonds between cysteine residues (see, e.g., thestructures depicted in FIGS. 1-20). In some embodiments, a sulfhydrylgroup on a cysteine residue can be replaced with an alternative group(e.g., —CH₂CH₂—). To replace a sulfhydryl group with a —CH₂— group, forexample, a cysteine residue can be replaced by alpha-aminobutyric acid.Such cyclic analog polypeptides can be generated, for example, asdescribed elsewhere (Lebl and Hruby, Tetrahedron Lett., 25:2067 (1984)and U.S. Pat. No. 4,161,521).

In some cases, ester or amide bridges can be formed by reacting the OHof serine or threonine with the carboxyl group of aspartic acid orglutamic acid to yield a bridge having the structure —CH₂CO₂CH₂—. Insome cases, an amide can be obtained by reacting the side chain oflysine with aspartic acid or glutamic acid to yield a bridge having thestructure —CH₂C(O)NH(CH)₄—. Methods for synthesis of these bridges aredescribed elsewhere (see, e.g., Schiller et al., Biochem. Biophy. Res.Comm., 127:558 (1985), and Schiller et al. Int. J. Peptide Protein Res.,25:171 (1985)). Other bridge-forming amino acid residues and reactionsare provided in, for example, U.S. Pat. No. 4,935,492. In some cases,peptide analogs that include non-peptidyl bonds can be used to linkamino acid residues of a chimeric polypeptide provided herein asdescribed elsewhere (See, e.g., Spatola et al., Life Sci., 38:1243(1986); Spatola, Vega Data, 1(3) (1983); Morley, Trends Pharm. Sci.,463-468 (1980); Hudson et al., Int. J. Pept. Prot. Res., 14:177 (1979);Spatola, in Chemistry and Biochemistry of Amino Acid Peptides andProteins, B. Weinstein, ed., Marcel Dekker, New York, p. 267 (1983);Hann, J. Chem. Soc. Perkin Trans., 1:307 (1982); Almquist et al., J.Med. Chem. 23:1392 (1980); Jennings-White et al., Tetrahedron Lett.,23:2533 (1982); European Patent Application EP 45665; Holladay et al.,Tetrahedron Lett., 24:4401 (1983); and Hruby, Life Sci., 31:189 (1982).

In some cases, a chimeric polypeptide provided herein can have an aminoacid sequence with at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identityto a reference sequence (e.g., SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61). Percentsequence identity is calculated by determining the number of matchedpositions in aligned amino acid sequences (target amino acid sequencealigned to an identified amino acid sequence), dividing the number ofmatched positions by the number of amino acids of the identified aminoacid sequence (e.g., SEQ ID NO:3), and multiplying by 100. A matchedposition refers to a position in which identical amino acids occur atthe same position in aligned amino acid sequences. Percent sequenceidentity also can be determined for any nucleic acid sequence.

Percent sequence identity is determined by comparing a target amino acidsequence to the identified amino acid sequence (e.g., SEQ ID NO:3) usingthe BLAST 2 Sequences (Bl2seq) program from the stand-alone version ofBLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14. Thisstand-alone version of BLASTZ can be obtained on the World Wide Web fromFish & Richardson's web site (fr.com/blast) or the U.S. government'sNational Center for Biotechnology Information web site(ncbi.nlm.nih.gov). Instructions explaining how to use the Bl2seqprogram can be found in the readme file accompanying BLASTZ.

Bl2seq performs a comparison between two sequences using either theBLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. Tocompare two nucleic acid sequences, the options are set as follows: -iis set to a file containing the first nucleic acid sequence to becompared (e.g., C:\seq1.txt); -j is set to a file containing the secondnucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set toblastn; -o is set to any desired file name (e.g., C:\output.txt); -q isset to −1; -r is set to 2; and all other options are left at theirdefault setting. The following command will generate an output filecontaining a comparison between two sequences: C:\Bl2seq-i c:\seq1.txt-jc:\seq2.txt-p blastn-o c:\output.txt-q−1-r 2. If the target sequenceshares homology with any portion of the identified sequence, then thedesignated output file will present those regions of homology as alignedsequences. If the target sequence does not share homology with anyportion of the identified sequence, then the designated output file willnot present aligned sequences.

For example, if (1) a target sequence is compared to the sequence setforth in SEQ ID NO:3 and (2) the Bl2seq program presents the targetsequence aligned with a region of the sequence set forth in SEQ ID NO:3with the number of matches being 15, then the amino acid target sequencehas a percent identity to SEQ ID NO:3 that is 88.2 (i.e.,15±17×100=88.2). It is noted that the percent identity value is roundedto the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 arerounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 arerounded up to 78.2. It also is noted that the length value will alwaysbe an integer.

A chimeric polypeptide provided herein can be produced using anysuitable method, including solid phase synthesis, and can be generatedusing manual techniques or automated techniques (e.g., using an AppliedBioSystems (Foster City, Calif.) Peptide Synthesizer or a Biosearch Inc.(San Rafael, Calif.) automatic peptide synthesizer). Disulfide bondsbetween cysteine residues can be introduced by mild oxidation of thelinear polypeptides using KCN as described elsewhere (U.S. Pat. No.4,757,048). In some cases, a chimeric polypeptide provided herein can beproduced recombinantly, as described herein.

In some cases, a chimeric polypeptide provided herein can be asubstantially pure polypeptide. As used herein, the term “substantiallypure” with reference to a polypeptide means that the polypeptide issubstantially free of other polypeptides, lipids, carbohydrates, andnucleic acid. In some cases, a substantially pure polypeptide can be apolypeptide that is at least 60 percent pure or is any chemicallysynthesized polypeptide. A substantially pure polypeptide can be atleast about 60, 65, 70, 75, 80, 85, 90, 95, or 99 percent pure.Typically, a substantially pure polypeptide will yield a single majorband on a non-reducing polyacrylamide gel.

Salts of carboxyl groups of a chimeric polypeptide provided herein canbe prepared by contacting the polypeptide with one or more equivalentsof a desired base such as, for example, a metallic hydroxide base (e.g.,sodium hydroxide), a metal carbonate or bicarbonate base (e.g., sodiumcarbonate or sodium bicarbonate), or an amine base (e.g., triethylamine,triethanolamine, and the like). Acid addition salts of a chimericpolypeptide provided herein can be prepared by contacting thepolypeptide with one or more equivalents of an inorganic or organic acid(e.g., hydrochloric acid).

Esters of carboxyl groups of a chimeric polypeptide provided herein canbe prepared using any suitable means for converting a carboxylic acid orprecursor to an ester. For example, one method for preparing esters of achimeric polypeptide provided herein, when using the Merrifieldsynthesis technique, is to cleave the completed polypeptide from theresin in the presence of the desired alcohol under either basic oracidic conditions, depending upon the resin. The C-terminal end of thepolypeptide then can be directly esterified when freed from the resin,without isolation of the free acid.

Amides of a chimeric polypeptide provided herein can be prepared usingtechniques for converting a carboxylic acid group or precursor to anamide. One method for amide formation at the C-terminal carboxyl groupincludes cleaving the polypeptide from a solid support with anappropriate amine, or cleaving in the presence of an alcohol, yieldingan ester, followed by aminolysis with the desired amine.

N-acyl derivatives of an amino group of a chimeric polypeptide providedherein can be prepared by utilizing an N-acyl protected amino acid forthe final condensation, or by acylating a protected or unprotectedpolypeptide. O-acyl derivatives can be prepared for example, byacylation of a free hydroxy peptide or peptide resin. Either acylationmay be carried out using standard acylating reagent such as acylhalides, anhydrides, acyl imidazoles, and the like. Both N- andO-acylation may be carried out together, if desired.

In some cases, a chimeric polypeptide provided herein can be modified bylinkage to a polymer such as polyethylene glycol (PEG), or by fusion toanother polypeptide such as albumin, for example. For example, one ormore PEG moieties can be conjugated to a chimeric polypeptide providedherein via lysine residues. Linkage to PEG or another suitable polymer,or fusion to albumin or another suitable polypeptide can result in amodified chimeric polypeptide having an increased half life as comparedto an unmodified chimeric polypeptide. Without being bound by aparticular mechanism, an increased serum half life can result fromreduced proteolytic degradation, immune recognition, or cell scavangingof the modified chimeric polypeptide. Any appropriate method can be usedto modify a chimeric polypeptide by linkage to PEG (also referred to as“PEGylation”) or other polymers including, without limitation, thosedescribed elsewhere (U.S. Pat. No. 6,884,780; Cataliotti et al., TrendsCardiovasc. Med., 17:10-14 (2007); Veronese and Mero, BioDrugs,22:315-329 (2008); Miller et al., Bioconjugate Chem., 17:267-274 (2006);and Veronese and Pasut, Drug Discov. Today, 10:1451-1458 (2005).Examples of methods for modifying a chimeric polypeptide by fusion toalbumin include, without limitation, those described elsewhere (U.S.Patent Publication No. 20040086976, and Wang et al., Pharm. Res.,21:2105-2111 (2004)).

The term “cardiac remodeling” refers to effects on the heart that canoccur with myocardial infarction, acute heart failure, or otherconditions. These include, for example, heart dilation, myocytehypertrophy, and cardiofibrosis (i.e., proliferation of interstitialfibroblasts). The chimeric polypeptides provided herein can be used toinhibit or prevent cardiac remodeling that occurs with myocardialinfarction or acute heart failure. In some cases, parameters indicativeof reduced cardiac remodeling can include one or more of the following:cardiac unloading (i.e., reduced pressure in the heart), increasedglomerular filtration rate (GFR), decreased plasma renin activity (PRA),decreased levels of angiotensin II, decreased proliferation of cardiacfibroblasts, decreased left ventricular (LV) hypertrophy, decreased LVmass (indicative of reduced fibrosis and hypertrophy), decreasedpulmonary capillary wedge pressure (PCWP; an indirect measure of leftatrial pressure), decreased right atrial pressure, decreased meanarterial pressure, decreased levels of aldosterone (indicative of ananti-fibrotic effect), decreased ventricular fibrosis, increasedejection fraction, and decreased LV end systolic diameter. To determinewhether a chimeric polypeptide provided herein is capable of inhibitingor reducing cardiac remodeling, one or more of these parameters can beevaluated (e.g., before and after treatment with the chimericpolypeptide), using methods known in the art and/or described herein,for example. The use of human amino acid sequences to construct achimeric polypeptide provided herein can minimize the risk ofimmunogenicity that may be observed with protein therapeutics, ascompared to the use of amino acid sequences from other species (Halleret al., Clin. Pharmacol. Ther, 84:624-7 (2008); and Leader et al., Nat.Rev. Drug Discov., 7:21-39 (2008)).

Conditions such as acute myocardial infarction (AMI) or acute heartfailure (AHF) can lead to kidney damage as well as heart damage. In somecases, the chimeric polypeptides provided herein can protect the kidneysfrom damage after AMI and AHF. Parameters that are indicative offavorable renal actions include, for example, decreased proximalfractional reabsorption of sodium (PFRNa), decreased distal fractionalreabsorption of sodium (DFRNa), increased urinary sodium excretion(UNaV), and increased urine flow (UV). Any one or more of theseparameters can be assessed (e.g., before and after administration of achimeric polypeptide) to determine whether the chimeric polypeptide haskidney protecting effects. Methods for assessing these parameters areknown in the art, and also are described herein.

In some cases, a chimeric polypeptide provided herein can inhibit orreduce cardiac remodeling such as occurs after AMI or AHF, for example.A chimeric polypeptide that can inhibit cardiac remodeling is one thatcan alter one or more parameters indicative of inhibited or reducedcardiac remodeling by at least 10%. To determine whether a particularchimeric polypeptide has such properties, one can carry out assays thatare well known to the art, including those described herein. A chimericpolypeptide that includes a variant NP sequence can have at least about10% (e.g., at least about 10%, 15%, 20%, 25%, 33%, 40%, 50%, 60%, 67%,75%, 80%, 85%, 90%, 95%, 100%, or more than 100%) of the biologicalactivity of the corresponding wild type NP sequence.

Nucleic Acids, Vectors, and Host Cells

This document also provides nucleic acids encoding a chimericpolypeptide provided herein, as well as expression vectors containingthe nucleic acids, and host cells containing the nucleic acids and/orexpression vectors. As used herein, the term “nucleic acid” refers toboth RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g.,chemically synthesized) DNA. A nucleic acid molecule can bedouble-stranded or single-stranded (i.e., a sense or an antisense singlestrand). Nucleic acids include, for example, cDNAs encoding the chimericpolypeptides provided herein.

An “isolated nucleic acid” is a nucleic acid that is separated fromother nucleic acid molecules that are present in a vertebrate genome,including nucleic acids that normally flank one or both sides of thenucleic acid in a vertebrate genome. The term “isolated” as used hereinwith respect to nucleic acids also includes any non-naturally-occurringnucleic acid sequence, since such non-naturally-occurring sequences arenot found in nature and do not have immediately contiguous sequences ina naturally-occurring genome.

An isolated nucleic acid can be, for example, a DNA molecule, providedone of the nucleic acid sequences normally found immediately flankingthat DNA molecule in a naturally-occurring genome is removed or absent.Thus, an isolated nucleic acid includes, without limitation, a DNAmolecule that exists as a separate molecule (e.g., a chemicallysynthesized nucleic acid, or a cDNA or genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences as well as DNA that is incorporated into a vector, anautonomously replicating plasmid, a virus (e.g., a retrovirus,lentivirus, adenovirus, or herpes virus), or into the genomic DNA of aprokaryote or eukaryote. In addition, an isolated nucleic acid caninclude an engineered nucleic acid such as a DNA molecule that is partof a hybrid or fusion nucleic acid. A nucleic acid existing amonghundreds to millions of other nucleic acids within, for example, cDNAlibraries or genomic libraries, or gel slices containing a genomic DNArestriction digest, is not considered an isolated nucleic acid.

Isolated nucleic acid molecules can be produced using standardtechniques, including, without limitation, common molecular cloning andchemical nucleic acid synthesis techniques. For example, polymerasechain reaction (PCR) techniques can be used to obtain an isolatednucleic acid containing a nucleotide sequence that encodes anangiotensin polypeptide or an NP. PCR refers to a procedure or techniquein which target nucleic acids are enzymatically amplified. Sequenceinformation from the ends of the region of interest or beyond typicallyis employed to design oligonucleotide primers that are identical insequence to opposite strands of the template to be amplified. PCR can beused to amplify specific sequences from DNA as well as RNA, includingsequences from total genomic DNA or total cellular RNA. Primerstypically are 14 to 40 nucleotides in length, but can range from 10nucleotides to hundreds of nucleotides in length. General PCR techniquesare described, for example in PCR Primer: A Laboratory Manual, ed. byDieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995.When using RNA as a source of template, reverse transcriptase can beused to synthesize complementary DNA (cDNA) strands. Ligase chainreaction, strand displacement amplification, self-sustained sequencereplication, or nucleic acid sequence-based amplification also can beused to obtain isolated nucleic acids. See, for example, Lewis (1992)Genetic Engineering News 12:1; Guatelli et al. (1990) Proc. Natl. Acad.Sci. USA 87:1874-1878; and Weiss (1991) Science 254:1292.

Isolated nucleic acids also can be chemically synthesized, either as asingle nucleic acid molecule (e.g., using automated DNA synthesis in the3′ to 5′ direction using phosphoramidite technology) or as a series ofoligonucleotides. For example, one or more pairs of longoligonucleotides (e.g., >100 nucleotides) can be synthesized thatcontain the desired sequence, with each pair containing a short segmentof complementarity (e.g., about 15 nucleotides) such that a duplex isformed when the oligonucleotide pair is annealed. DNA polymerase is usedto extend the oligonucleotides, resulting in a single, double-strandednucleic acid molecule per oligonucleotide pair, which then can beligated into a vector.

Isolated nucleic acids (e.g., nucleic acids encoding variant NPs) alsocan be obtained by mutagenesis. For example, a reference sequence can bemutated using standard techniques including oligonucleotide-directedmutagenesis and site-directed mutagenesis through PCR. See, ShortProtocols in Molecular Biology, Chapter 8, Green Publishing Associatesand John Wiley & Sons, edited by Ausubel et al., 1992. Non-limitingexamples of variant NPs art provided herein.

Sources of nucleotide sequences from which nucleic acid moleculesencoding an NP, or the nucleic acid complement thereof, can be obtainedinclude total or polyA⁺ RNA from any eukaryotic source, includingreptilian (e.g., snake) or mammalian (e.g., human, rat, mouse, canine,bovine, equine, ovine, caprine, or feline) cellular source from whichcDNAs can be derived by methods known in the art. Other sources of thenucleic acid molecules include genomic libraries derived from anyeukaryotic cellular source, including mammalian sources.

Nucleic acid molecules encoding native NPs can be identified andisolated using standard methods, e.g., as described by Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, NY (1989). For example, reverse-transcriptase PCR (RT-PCR) can beused to isolate and clone NP cDNAs from isolated RNA that contains RNAsequences of interest (e.g., total RNA isolated from human tissue).Other approaches to identify, isolate, and clone NP cDNAs include, forexample, screening cDNA libraries.

Vectors containing nucleic acids such as those described herein also areprovided. A “vector” is a replicon, such as a plasmid, phage, or cosmid,into which another DNA segment may be inserted so as to bring about thereplication of the inserted segment. An “expression vector” is a vectorthat includes one or more expression control sequences, and an“expression control sequence” is a DNA sequence that controls andregulates the transcription and/or translation of another DNA sequence.

In the expression vectors, a nucleic acid (e.g., a nucleic acid encodinga chimeric polypeptide provided herein) can be operably linked to one ormore expression control sequences. As used herein, “operably linked”means incorporated into a genetic construct so that expression controlsequences effectively control expression of a coding sequence ofinterest. Examples of expression control sequences include promoters,enhancers, and transcription terminating regions. A promoter is anexpression control sequence composed of a region of a DNA molecule,typically within 100 to 500 nucleotides upstream of the point at whichtranscription starts (generally near the initiation site for RNApolymerase II). To bring a coding sequence under the control of apromoter, it is necessary to position the translation initiation site ofthe translational reading frame of the polypeptide between one and aboutfifty nucleotides downstream of the promoter. Enhancers provideexpression specificity in terms of time, location, and level. Unlikepromoters, enhancers can function when located at various distances fromthe transcription site. An enhancer also can be located downstream fromthe transcription initiation site. A coding sequence is “operablylinked” and “under the control” of expression control sequences in acell when RNA polymerase is able to transcribe the coding sequence intomRNA, which then can be translated into the protein encoded by thecoding sequence.

Suitable expression vectors include, without limitation, plasmids andviral vectors derived from, for example, bacteriophage, baculoviruses,tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses,vaccinia viruses, adenoviruses, and adeno-associated viruses. Numerousvectors and expression systems are commercially available from suchcorporations as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.),Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies(Carlsbad, Calif.).

An expression vector can include a tag sequence designed to facilitatesubsequent manipulation of the expressed nucleic acid sequence (e.g.,purification or localization). Tag sequences, such as green fluorescentprotein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc,hemagglutinin, or Flag™ tag (Kodak, New Haven, Conn.) sequencestypically are expressed as a fusion with the encoded polypeptide. Suchtags can be inserted anywhere within the polypeptide including at eitherthe carboxyl or amino terminus.

This document also provides host cells containing a nucleic acid orvector provided herein. The term “host cell” is intended to includeprokaryotic and eukaryotic cells into which a recombinant nucleic acidor vector (e.g., an expression vector) can be introduced. As usedherein, “transformed” and “transfected” encompass the introduction of anucleic acid molecule (e.g., a vector) into a cell by one of a number oftechniques. Although not limited to a particular technique, a number ofthese techniques are well established within the art. Suitable methodsfor transforming and transfecting host cells can be found, for example,in Sambrook et al., Molecular Cloning: A Laboratory Manual (2^(nd)edition), Cold Spring Harbor Laboratory, New York (1989). For example,calcium phosphate precipitation, electroporation, heat shock,lipofection, microinjection, and viral-mediated nucleic acid transfercan be used introduce nucleic acid into cells. In addition, naked DNAcan be delivered directly to cells in vivo as described elsewhere (U.S.Pat. Nos. 5,580,859 and 5,589,466).

Detecting Polypeptides

This document also provides methods and materials for detecting achimeric polypeptide provided herein. Such methods and materials can beused to monitor chimeric polypeptide levels within a mammal receivingthe chimeric polypeptide as a therapeutic. A chimeric polypeptideprovided herein (e.g., a chimeric polypeptide as set forth in any one ofFIGS. 2-20 and 23-32) can be detected, for example, immunologicallyusing one or more antibodies. As used herein, the term “antibody”includes intact molecules as well as fragments thereof that are capableof binding to an epitopic determinant of a chimeric polypeptide providedherein. The term “epitope” refers to an antigenic determinant on anantigen to which the paratope of an antibody binds. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains, and typically havespecific three-dimensional structural characteristics, as well asspecific charge characteristics. Epitopes generally have at least fivecontiguous amino acids (a continuous epitope), or alternatively can be aset of noncontiguous amino acids that define a particular structure(e.g., a conformational epitope). The term “antibody” includespolyclonal antibodies, monoclonal antibodies, humanized or chimericantibodies, single chain Fv antibody fragments, Fab fragments, andF(ab)₂ fragments. Polyclonal antibodies are heterogeneous populations ofantibody molecules that are contained in the sera of the immunizedanimals. Monoclonal antibodies are homogeneous populations of antibodiesto a particular epitope of an antigen.

Antibody fragments that have specific binding affinity for a chimericpolypeptide provided herein (e.g., a chimeric polypeptide as set forthin any one of FIGS. 2-20 and 23-32) can be generated by knowntechniques. For example, F(ab′)2 fragments can be produced by pepsindigestion of the antibody molecule, and Fab fragments can be generatedby reducing the disulfide bridges of F(ab′)2 fragments. In some cases,Fab expression libraries can be constructed. See, for example, Huse etal., Science, 246:1275 (1989). Once produced, antibodies or fragmentsthereof can be tested for recognition of a chimeric polypeptide providedherein by standard immunoassay methods including ELISA techniques,radioimmunoassays, and Western blotting. See, Short Protocols inMolecular Biology, Chapter 11, Green Publishing Associates and JohnWiley & Sons, Ed. Ausubel et al., 1992.

In immunological assays, an antibody having specific binding affinityfor a chimeric polypeptide provided herein or a secondary antibody thatbinds to such an antibody can be labeled, either directly or indirectly.Suitable labels include, without limitation, radionuclides (e.g., ¹²⁵I,¹³¹I, ³⁵S, ³H, ³²P, ³³P, or ¹⁴C), fluorescent moieties (e.g.,fluorescein, FITC, PerCP, rhodamine, or PE), luminescent moieties (e.g.,Qdot™ nanoparticles supplied by Invitrogen (Carlsbad, Calif.)),compounds that absorb light of a defined wavelength, or enzymes (e.g.,alkaline phosphatase or horseradish peroxidase). Antibodies can beindirectly labeled by conjugation with biotin then detected with avidinor streptavidin labeled with a molecule described above. Methods ofdetecting or quantifying a label depend on the nature of the label andare known in the art. Examples of detectors include, without limitation,x-ray film, radioactivity counters, scintillation counters,spectrophotometers, colorimeters, fluorometers, luminometers, anddensitometers. Combinations of these approaches (including “multi-layer”assays) familiar to those in the art can be used to enhance thesensitivity of assays.

Immunological assays for detecting a polypeptide provided herein can beperformed in a variety of known formats, including sandwich assays,competition assays (competitive RIA), or bridge immunoassays. See, forexample, U.S. Pat. Nos. 5,296,347; 4,233,402; 4,098,876; and 4,034,074.Methods of detecting a chimeric polypeptide provided herein generallyinclude contacting a biological sample with an antibody that binds to achimeric polypeptide provided herein and detecting binding of thechimeric polypeptide to the antibody. For example, an antibody havingspecific binding affinity for a chimeric polypeptide provided herein canbe immobilized on a solid substrate by any of a variety of methods knownin the art and then exposed to the biological sample. Binding of thechimeric polypeptide to the antibody on the solid substrate can bedetected by exploiting the phenomenon of surface plasmon resonance,which results in a change in the intensity of surface plasmon resonanceupon binding that can be detected qualitatively or quantitatively by anappropriate instrument, e.g., a Biacore apparatus (Biacore InternationalAB, Rapsgatan, Sweden). In some cases, the antibody can be labeled anddetected as described above. A standard curve using known quantities ofa chimeric polypeptide provided herein can be generated to aid in thequantitation of the levels of the chimeric polypeptide.

In some embodiments, a “sandwich” assay in which a capture antibody isimmobilized on a solid substrate can be used to detect the presence,absence, or level of a chimeric polypeptide provided herein. The solidsubstrate can be contacted with the biological sample such that anychimeric polypeptide of interest in the sample can bind to theimmobilized antibody. The presence, absence, or level of the chimericpolypeptide bound to the antibody can be determined using a “detection”antibody having specific binding affinity for the chimeric polypeptide.In some embodiments, a capture antibody can be used that has bindingaffinity for ANP, BNP, CNP, DNP, URO, or an angiotensin polypeptide aswell as a chimeric polypeptide provided herein. In this embodiment, adetection antibody can be used that has specific binding affinity for aparticular chimeric polypeptide provided herein (e.g., a chimericpolypeptide as set forth in any one of FIGS. 2-20 and 23-32). It isunderstood that in sandwich assays, the capture antibody should not bindto the same epitope (or range of epitopes in the case of a polyclonalantibody) as the detection antibody. Thus, if a monoclonal antibody isused as a capture antibody, the detection antibody can be anothermonoclonal antibody that binds to an epitope that is either physicallyseparated from or only partially overlaps with the epitope to which thecapture monoclonal antibody binds, or a polyclonal antibody that bindsto epitopes other than or in addition to that to which the capturemonoclonal antibody binds. If a polyclonal antibody is used as a captureantibody, the detection antibody can be either a monoclonal antibodythat binds to an epitope that is either physically separated from orpartially overlaps with any of the epitopes to which the capturepolyclonal antibody binds, or a polyclonal antibody that binds toepitopes other than or in addition to that to which the capturepolyclonal antibody binds. Sandwich assays can be performed as sandwichELISA assays, sandwich Western blotting assays, or sandwichimmunomagnetic detection assays.

Suitable solid substrates to which an antibody (e.g., a captureantibody) can be bound include, without limitation, microtiter plates,tubes, membranes such as nylon or nitrocellulose membranes, and beads orparticles (e.g., agarose, cellulose, glass, polystyrene, polyacrylamide,magnetic, or magnetizable beads or particles). Magnetic or magnetizableparticles can be particularly useful when an automated immunoassaysystem is used.

Antibodies having specific binding affinity for a chimeric polypeptideprovided herein can be produced through standard methods. For example, achimeric polypeptide can be recombinantly produced as described above orcan be chemically synthesized, and used to immunize host animals,including rabbits, chickens, mice, guinea pigs, or rats. For example, achimeric polypeptide as set forth in any one of FIGS. 2-20 and 23-32 canbe used to immunize an animal. Various adjuvants that can be used toincrease the immunological response depend on the host species andinclude Freund's adjuvant (complete and incomplete), mineral gels suchas aluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin and dinitrophenol. Monoclonal antibodies can be preparedusing a chimeric polypeptide provided herein and standard hybridomatechnology. In particular, monoclonal antibodies can be obtained by anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture such as described by Kohler et al.,Nature, 256:495 (1975), the human B-cell hybridoma technique (Kosbor etal., Immunology Today, 4:72 (1983); Cole et al., Proc. Natl. Acad. Sci.USA, 80:2026 (1983)), and the EBV-hybridoma technique (Cole et al.,“Monoclonal Antibodies and Cancer Therapy,” Alan R. Liss, Inc., pp.77-96 (1983)). Such antibodies can be of any immunoglobulin classincluding IgG, IgM, IgE, IgA, IgD, and any subclass thereof. Thehybridoma producing the monoclonal antibodies can be cultivated in vitroand in vivo.

In some cases, antibodies directed to either an NP sequence or thesequence of an angiotensin polypeptide or a combination of both types ofantibodies can be used to detect a chimeric polypeptide provided herein(e.g., a chimeric polypeptide as set forth in any one of FIGS. 2-20 and23-32).

Other techniques for detecting a chimeric polypeptide provided hereininclude mass-spectrophotometric techniques such as electrosprayionization (ESI), and matrix-assisted laser desorption-ionization(MALDI). See, for example, Gevaert et al., Electrophoresis, 22:1645-51(2001); Chaurand et al., J. Am. Soc. Mass Spectrom., 10:91-103 (1999).Mass spectrometers useful for such applications are available fromApplied Biosystems (Foster City, Calif.); Bruker Daltronics (Billerica,Mass.); and Amersham Pharmacia (Sunnyvale, Calif.).

Compositions and Methods for Administration

A chimeric polypeptide provided herein (e.g., a chimeric polypeptide asset forth in any one of FIGS. 2-20 and 23-32), or a nucleic acidencoding a chimeric polypeptide provided herein, can be incorporatedinto a composition for administration to a mammal (e.g., a humansuffering from or at risk for AMI or AHF). Methods for formulating andsubsequently administering therapeutic compositions are well known tothose in the art. Dosages typically are dependent on the responsivenessof the subject to the compound, with the course of treatment lastingfrom several days to several months, or until a suitable response isachieved. Persons of ordinary skill in the art routinely determineoptimum dosages, dosing methodologies, and repetition rates. Optimumdosages can vary depending on the relative potency of a chimericpolypeptide, and generally can be estimated based on the EC₅₀ found tobe effective in in vitro and/or in vivo animal models. Compositionscontaining a chimeric polypeptide provided herein or a nucleic acidprovided herein may be given once or more daily, weekly, monthly, oreven less often, or can be administered continuously for a period oftime (e.g., hours, days, or weeks). For example, a chimeric polypeptideprovided herein or a composition containing a chimeric polypeptideprovided herein can be administered to an myocardial infarction patientat a dose of at least about 0.01 ng polypeptide/kg to about 100 mgpolypeptide/kg of body mass at or about the time of reperfusion, or canbe administered continuously as an infusion beginning at or about thetime of reperfusion and continuing for one to seven days (e.g., at adose of about 0.01 ng polypeptide/kg/minute to about 0.5 μgpolypeptide/kg/minute).

The chimeric polypeptides or nucleic acids can be admixed, encapsulated,conjugated or otherwise associated with other molecules, molecularstructures, or mixtures of compounds such as, for example, liposomes,receptor or cell targeted molecules, or oral, topical or otherformulations for assisting in uptake, distribution and/or absorption.

In some embodiments, a composition can contain a chimeric polypeptideprovided herein in combination with a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers include, for example,pharmaceutically acceptable solvents, suspending agents, or any otherpharmacologically inert vehicles for delivering antibodies to a subject.Pharmaceutically acceptable carriers can be liquid or solid, and can beselected with the planned manner of administration in mind so as toprovide for the desired bulk, consistency, and other pertinent transportand chemical properties, when combined with one or more therapeuticcompounds and any other components of a given pharmaceuticalcomposition. Typical pharmaceutically acceptable carriers include,without limitation: water; saline solution; binding agents (e.g.,polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose or dextrose and other sugars, gelatin, or calcium sulfate);lubricants (e.g., starch, polyethylene glycol, or sodium acetate);disintegrates (e.g., starch or sodium starch glycolate); and wettingagents (e.g., sodium lauryl sulfate).

Pharmaceutical compositions containing a chimeric polypeptide providedherein can be administered by a number of methods, depending uponwhether local or systemic treatment is desired. Administration can be,for example, parenteral (e.g., by subcutaneous, intrathecal,intraventricular, intramuscular, or intraperitoneal injection, or byintravenous (i.v.) drip); oral; topical (e.g., transdermal, sublingual,ophthalmic, or intranasal); or pulmonary (e.g., by inhalation orinsufflation of powders or aerosols), or can occur by a combination ofsuch methods. Administration can be rapid (e.g., by injection) or canoccur over a period of time (e.g., by slow infusion or administration ofslow release formulations).

Compositions and formulations for parenteral, intrathecal orintraventricular administration include sterile aqueous solutions (e.g.,sterile physiological saline), which also can contain buffers, diluentsand other suitable additives (e.g., penetration enhancers, carriercompounds and other pharmaceutically acceptable carriers).

Compositions and formulations for oral administration include, forexample, powders or granules, suspensions or solutions in water ornon-aqueous media, capsules, sachets, or tablets. Such compositions alsocan incorporate thickeners, flavoring agents, diluents, emulsifiers,dispersing aids, or binders.

Pharmaceutical compositions include, but are not limited to, solutions,emulsions, aqueous suspensions, and liposome-containing formulations.These compositions can be generated from a variety of components thatinclude, for example, preformed liquids, self-emulsifying solids andself-emulsifying semisolids. Emulsion formulations are particularlyuseful for oral delivery of therapeutic compositions due to their easeof formulation and efficacy of solubilization, absorption, andbioavailability. Liposomes can be particularly useful due to theirspecificity and the duration of action they offer from the standpoint ofdrug delivery.

Compositions additionally can contain other adjunct componentsconventionally found in pharmaceutical compositions. Thus, thecompositions also can include compatible, pharmaceutically activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or additional materials usefulin physically formulating various dosage forms of the compositions, suchas dyes, flavoring agents, preservatives, antioxidants, opacifiers,thickening agents, and stabilizers. Furthermore, the composition can bemixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, colorings, flavorings, penetration enhancers, andaromatic substances. When added, however, such materials should notunduly interfere with the biological activities of the other componentswithin the compositions (e.g., a chimeric polypeptide provided herein).

In some cases, a chimeric polypeptide provided herein can be formulatedas a sustained release dosage form. In some cases, coatings, envelopes,or protective matrices can be formulated to contain one or more of thechimeric polypeptides provided herein. Such coatings, envelopes, andprotective matrices can be used to coat indwelling devices such asstents, catheters, and peritoneal dialysis tubing. In some cases, achimeric polypeptide provided herein can be incorporated into apolymeric substances, liposomes, microemulsions, microparticles,nanoparticles, or waxes.

Pharmaceutical formulations as disclosed herein, which can be presentedconveniently in unit dosage form, can be prepared according toconventional techniques well known in the pharmaceutical industry. Suchtechniques include the step of bringing into association an activeingredient (e.g., a chimeric polypeptide provided herein) with thedesired pharmaceutical carrier(s). Typically, the formulations can beprepared by uniformly and intimately bringing an active ingredient intoassociation with liquid carriers or finely divided solid carriers orboth, and then, if necessary, shaping the product. Formulations can besterilized if desired, provided that the method of sterilization doesnot interfere with the effectiveness of the molecules(s) contained inthe formulation (e.g., a chimeric polypeptide provided herein).

Methods for Reducing or Inhibiting Cardiac Remodeling

This document also provides for the use of a chimeric polypeptideprovided herein for treatment of, for example, AHF and AMI. For example,a chimeric polypeptide provided herein can be administered to a mammal(e.g., a human or a non-human mammal) in order to reduce or inhibitcardiac remodeling that can occur, for example, after myocardialinfarction. In some embodiments, for example, a chimeric polypeptideprovided herein or a composition provided herein can be administered toa mammal diagnosed as having had an AMI. The chimeric polypeptide orcomposition can be administered at any suitable dose, depending onvarious factors including, without limitation, the agent chosen, thedisease, and whether prevention or treatment is to be achieved.Administration can be local or systemic.

In some embodiments, a chimeric polypeptide provided herein or acomposition containing a chimeric polypeptide provided herein can beadministered at a dose of at least about 0.01 ng polypeptide/kg to about100 mg polypeptide/kg of body mass (e.g., about 10 ng polypeptide/kg toabout 50 mg polypeptide/kg, about 20 ng polypeptide/kg to about 10 mgpolypeptide/kg, about 0.1 ng polypeptide/kg to about 20 ngpolypeptide/kg, about 3 ng polypeptide/kg to about 10 ng polypeptide/kg,or about 50 ng polypeptide/kg to about 100 μg/kg) of body mass, althoughother dosages also may provide beneficial results. In some cases, achimeric polypeptide provided herein or a composition containing achimeric polypeptide provided herein can be administered as a continuousintravenous infusion beginning at or about the time of reperfusion(i.e., at the time the occluded artery is opened), and continuing forone to seven days (e.g., one, two, three, four, five, six, or sevendays). Such a composition can be administered at a dose of, for example,about 0.1 ng polypeptide/kg/minute to about 500 ng polypeptide/kg/minute(e.g., about 0.5 ng polypeptide/kg/minute, about 1 ngpolypeptide/kg/minute, about 2 ng polypeptide/kg/minute, about 3 ngpolypeptide/kg/minute, about 5 ng polypeptide/kg/minute, about 7.5 ngpolypeptide/kg/minute, about 10 ng polypeptide/kg/minute, about 12.5 ngpolypeptide/kg/minute, about 15 ng polypeptide/kg/minute, about 20 ngpolypeptide/kg/minute, about 25 ng polypeptide/kg/minute, about 30 ngpolypeptide/kg/minute, about 50 ng polypeptide/kg/minute, about 100 ngpolypeptide/kg/minute, or about 300 ng polypeptide/kg/minute). In someembodiments, a chimeric polypeptide provided herein or a compositioncontaining a chimeric polypeptide provided herein can be administeredbefore reperfusion (e.g., about one hour prior to reperfusion), eitheras one or more individual doses or as a continuous infusion beginningabout one hour prior to reperfusion). For example, a composition can beadministered beginning about one hour, about 45 minutes, about 30minutes, or about 15 minutes prior to reperfusion. In some cases, achimeric polypeptide provided herein or a composition containing achimeric polypeptide provided herein can be administered afterreperfusion (e.g., within about ten hours of reperfusion), and can beadministered either as one or more individual doses or as a continuousinfusion beginning within about ten hours of reperfusion. For example, achimeric polypeptide provided herein or a composition containing achimeric polypeptide provided herein can be administered about one hour,about two hours, about three hours, about four hours, about five hours,about six hours, about seven hours, about eight hours, about nine hours,or about ten hours after reperfusion.

In some embodiments, a chimeric polypeptide provided herein or acomposition containing a chimeric polypeptide provided herein can beadministered via a first route (e.g., intravenously) for a first periodof time, and then can be administered via another route (e.g., topicallyor subcutaneously) for a second period of time. For example, a chimericpolypeptide provided herein or a composition containing a chimericpolypeptide provided herein can be intravenously administered to amammal (e.g., a human) at a dose of about 0.1 ng polypeptide/kg/minuteto about 300 ng polypeptide/kg/minute (e.g., about 1 ngpolypeptide/kg/minute to about 15 ng polypeptide/kg/minute, about 3 ngpolypeptide/kg/minute to about 10 ng polypeptide/kg/minute, or about 10ng polypeptide/kg/minute to about 30 ng polypeptide/kg/minute) for oneto seven days (e.g., one, two, three, four, five, six, or seven days),and subsequently can be subcutaneously administered to the mammal at adose of about 10 ng polypeptide/kg/day to about 100 ngpolypeptide/kg/day (e.g., about 10 ng polypeptide/kg/day, about 20 ngpolypeptide/kg/day, about 25 ng polypeptide/kg/day, about 30 ngpolypeptide/kg/day, about 50 ng polypeptide/kg/day, or about 100 ngpolypeptide/kg/day) for five to 30 days (e.g., seven, 10, 14, 18, 21,24, or 27 days).

The methods provided herein can include administering to a mammal aneffective amount of a chimeric polypeptide provided herein or acomposition containing a chimeric polypeptide provided herein. As usedherein, the term “effective amount” is an amount of a molecule orcomposition that is sufficient to alter one or more (e.g., one, two,three, four, five, six, seven, eight, nine, or ten) parametersindicative of reduced cardiac remodeling and/or kidney protection in amammalian recipient by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 40%,50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%). For example, aneffective amount of a chimeric polypeptide provided herein or acomposition containing a chimeric polypeptide provided herein is anamount that can increase ejection fraction, GFR, urinary sodiumexcretion (UNaV), or urine flow (UV) by at least 10%, and/or that candecrease PRA, LV mass, CF proliferation, PCWP, RAP, MAP, aldosteronelevels, LV hypertrophy, ventricular fibrosis, LV end systolic diameter,PFRNa, or DFRNa by at least 10%, and/or that can result in cardiacunloading. In some embodiments, a method can include administering to amammal an amount of a chimeric polypeptide provided herein or acomposition containing a chimeric polypeptide provided herein that issufficient to alter one or more parameters indicative of reduced cardiacremodeling and/or kidney protection by at least 50%.

In some embodiments, for example, an “effective amount” of a chimericpolypeptide provided herein or a composition containing a chimericpolypeptide provided herein can be an amount that reduces PRA and MAPand increases GFR and UV in a treated mammal by at least 10% as comparedto the levels of those parameters in the mammal prior to administrationof the chimeric polypeptide or composition or without administration ofthe chimeric polypeptide or composition (e.g., the level of theparameters observed in a previous myocardial infarction episode).

The invention will be further described in the following example, whichdoes not limit the scope of the invention described in the claims.

EXAMPLE Example 1 cANG Induce cGMP Production

cANG was custom-synthesized to have the sequence set forth in FIG. 4 andconfirmed by high performance liquid chromatography and massspectroscopy to have a molecular weight of 3080.6 Da. Human cardiacfibroblasts (CFs; ScienCell, San Diego, Calif.) were cultured in themanufacturer's fibroblast media (ScienCell, San Diego, Calif.)supplemented with fibroblast growth serum (FGS), fetal bovine serum(FBS), and Pen/Strep. Cells were treated at 80-90% confluency. Only cellpassages 1 through 4 were used for experiments. To perform a cyclic GMPassay, the cells were treated as described previously (Huntley et al.,J. Cell. Physiol., 209(3):943-9 (2006)). Briefly, cells were incubatedin Hank's balanced salt solution (Invitrogen, Carlsbad, Calif.)containing 20 mmol/L N-[2-hydroxyethyl]piperazine-N′[2-ethanesulfonicacid], 0.1% bovine serum albumin, and 0.5 mmol/L3-isobutyl-1-methylzanthine (Sigma, St. Louis, Mo.). Treated cellsreceived 10⁻⁶ M, 10⁻⁸ M, or 10⁻¹⁰ M of NP or cANG for 10 minutes. Cellswere lysed in 6% TCA and sonicated for 10 minutes. The samples wereether extracted four times in four volumes of ether, dried, andreconstituted in 500 ml of cGMP assay buffer.

The samples were assayed using a competitive RIA cGMP kit (Perkin-Elmer,Boston, Mass.). Briefly, samples and standards were incubated with 100mL anti-human cGMP polyclonal antibody and I¹²⁵-antigen for 18 hours.Cyclic GMP assay buffer was added to the samples, and they werecentrifuged for 20 minutes at 2500 rpm. The free fraction was aspiratedoff, and the bound fraction was counted and concentrations determined.Samples were corrected for dilution factors and protein concentration,and values were expressed as pmoles/mL. The assay was highly specificfor cGMP, demonstrating no cross-reactivity with ANP, BNP, CNP, andEndothelin-1, and less than 0.001 percent cross-reactivity with cAMP,GMP, GDP, ATP, and GTP.

Like the treatment of CFs with ANP, BNP, and CNP, treatment of CFs withcANG resulted in cGMP production (FIGS. 20 and 21). These resultsdemonstrate that addition of Ang1-7 does not interfere with receptorbinding and cGMP generation.

Example 2 BNP-Ang1-7 and Ang1-7BNP Induce cGMP Production

BNP-Ang1-7 and Ang1-7BNP were synthesized to have the sequence set forthin FIG. 3 and FIG. 20, respectively.

HEK 293 cells were stably transfected to express either NPR-A (GC-A) orNPR-B (GC-B) using Lipofectamine (Invitrogen, Grand Island, N.Y.).Transfected cells were maintained in Dulbecco's modified Eagle's mediumsupplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 U/mLstreptomycin, and 250 μg/mL G418. The reagents were obtained fromInvitrogen (Grand Island, N.Y.).

The following was performed to carry out cell stimulation and cGMPassays. Cells were plated in 6-well plates and treated as describedelsewhere (Tsuruda et al., Circulation Research, 91:1127-1134 (2002)).Briefly, cells were incubated in Hank's balanced salt solution(Invitrogen, Carlsbad, Calif.) containing 20 mmol/LN-[2-hydroxyethyl]piperazine-N′[2-ethanesulfonic acid], 0.1% bovineserum albumin, and 0.5 mmol/L 3-isobutyl-1-methylzanthine (Sigma, St.Louis, Mo.). Treated cells received 10⁻¹⁰ M, 10⁻⁸ M, or 10⁻⁶ M of eitherBNP-Ang1-7 or Ang1-7BNP for 10 minutes. Cells were lysed in 300 μL 6%TCA and sonicated for 10 minutes. The samples were ether extracted fourtimes in 4 volumes of ether, dried, and reconstituted in 500 μL cGMPassay buffer. The samples were assayed using a competitive RIA cGMP kit(Perkin-Elmer, Boston, Mass.) as described elsewhere (Steiner et al., J.Biol. Chem., 247:1106-1113 (1972)). Samples were corrected for dilutionfactors and protein concentration, and values were expressed as pmolcGMP/well. There was no cross-reactivity with ANP, BNP, CNP, andEndothelin-1, and less than 0.001 percent cross-reactivity with cAMP,GMP, GDP, ATP, and GTP.

Like BNP (FIG. 33A), exposure of NPR-A⁺ HEK 293 cells to eitherBNP-Ang1-7 or Ang1-7BNP induced cGMP production (FIGS. 33B and 33C).Neither BNP-Ang1-7 nor Ang1-7BNP induced cGMP production in NPR-B⁺ HEK293 cells (FIGS. 34B and 34C). Minimal cGMP production was observed forNPR-B⁺ HEK 293 cells exposed to BNP (FIG. 34A). Ang1-7 did not inducecGMP production when placed in contact with NPR-A⁺ HEK 293 cells (FIG.35, left panel) or NPR-B⁺ HEK 293 cells (FIG. 35, right panel).

These results demonstrate that both BNP-Ang1-7 and Ang1-7BNP induce cGMPproduction. These results also demonstrate that attaching Ang1-7 to anatriuretic polypeptide or a component of a natriuretic polypeptide(e.g., a ring structure of a natriuretic polypeptide) in either anN-terminal location or a C-terminal location does not interfere with theability of the natriuretic polypeptide or the natriuretic polypeptidecomponent to bind to its natriuretic polypeptide receptor and inducecGMP production.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A polypeptide from 24 to 55 amino acid residuesin length, wherein said polypeptide comprises, in an order from aminoterminus to carboxy terminus: (a) the sequence set forth in SEQ ID NO:1or the sequence set forth in SEQ ID NO:1with no more than two additions,deletions, or substitutions, (b) the sequence set forth in SEQ ID NO:3or the sequence set forth in SEQ ID NO: 3 with no more than twoadditions, deletions, or substitutions, and (c) the sequence set forthin SEQ ID NO:12 or the sequence set forth in SEQ ID NO: 12 with no morethan two additions, deletions, or substitutions.
 2. The polypeptide ofclaim 1, wherein said polypeptide comprises the sequence set forth inSEQ ID NO:1 with two additions, deletions, or substitutions.
 3. Thepolypeptide of claim 1, wherein said polypeptide comprises the sequenceset forth in SEQ ID NO:1 with one addition, deletion, or substitution.4. The polypeptide of claim 1, wherein said polypeptide comprises thesequence set forth in SEQ ID NO:1.
 5. The polypeptide of claim 1,wherein said polypeptide comprises the sequence set forth in SEQ ID NO:3with two additions, deletions, or substitutions.
 6. The polypeptide ofclaim 1, wherein said polypeptide comprises the sequence set forth inSEQ ID NO:3 with one addition, deletion, or substitution.
 7. Thepolypeptide of claim 1, wherein said polypeptide comprises the sequenceset forth in SEQ ID NO:3.
 8. The polypeptide of claim 1, wherein saidpolypeptide comprises the sequence set forth in SEQ ID NO:12 with twoadditions, deletions, or substitutions.
 9. The polypeptide of claim 1,wherein said polypeptide comprises the sequence set forth in SEQ IDNO:12 with one addition, deletion, or substitution.
 10. The polypeptideof claim 1, wherein said polypeptide comprises the sequence set forth inSEQ ID NO:12.
 11. The polypeptide of claim 1, wherein said polypeptidecomprises the sequence set forth in SEQ ID NO:1, the sequence set forthin SEQ ID NO:3, and the sequence set forth in SEQ ID NO:12.
 12. Thepolypeptide of claim 1, wherein said polypeptide is as set forth in SEQID NO:60.